1. Field of the Invention
The present invention generally concerns the field of virology, in particular the field of viral vaccine development.
2. Description of Related Art
Flaviviruses are a genus of blood borne pathogens that pose a significant threat to human. Flaviviruses include a variety of human pathogens such as West Nile (WNV), yellow fever (YF) and dengue (DEN) viruses. The flavivirus genome is a single-stranded, positive-sense RNA molecule approximately 11 kb in length encoding a single polyprotein that is co- and post-translationally cleaved by a combination of viral and host proteases to produce three structural and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).
In the United States West Nile virus has recently become a major human heath concern. West Nile virus is a member of the Japanese encephalitis (JE) serogroup, which also comprises Murray Valley encephalitis (MVE) and JE viruses, and was first isolated in the West Nile region of Uganda in 1937 (Smithburn 1940). Until recently WNV was found only in Africa, Asia, and Europe but emerged in the New World in 1999 when it was identified in New York. Since its introduction into northeastern U.S., WNV has spread throughout North America and has been responsible for over 16,000 human cases and 550 deaths (MMWR). There are a range of disease manifestations caused by WNV from inapparent infection to encephalitis and death due to the potential neuroinvasive and neurovirulence phenotypes of the virus.
Like other flaviviruses the WNV genome consists of a single open reading frame which encodes three structural genes including the capsid (C), membrane (prM/M), and envelope (E), 7 nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and is flanked by 5′ and 3′ noncoding regions. Flaviviruses are unusual RNA viruses in that the NS1 protein is glycosylated in addition to the E protein (Muylaert 1990). The NS1 protein may have either two or three highly conserved glycosylation sites. All members of the JE serogroup, with the exception of JE virus, contain three glycosylation sites in the NS1 protein at positions NS1130, NS1175, and NS1207 (Chambers 1990; Blitvich 2001, Sumiyoshi 1987). Other mosquito-borne flaviviruses, including JE and DEN viruses, contain two glycosylation sites in the NS1 protein at positions NS1130 and NS1207, while YF virus includes two sites at positions NS1130 and NS1208. Although the functions of the NS1 protein have not been completely elucidated, previous studies have shown that NS1 is involved in replication as shown by the colocalization of this protein and other NS proteins to double stranded RNA replicative forms (Mackenzie 1996; Westaway 1997), maturation of the virus (Mackenzie 1996) and RNA synthesis (Lindenbach and Rice 1997). Recently it has been noted that the NS1 protein may mimic extracellular matrix proteins and function to induce autoreactive antibodies (Falconar 1997, Chang 2002).
The NS1 protein exists as a hexamer, heat-labile homodimer or short-lived monomer and can be found inside the cell, associated with membranes or in secreted forms outside of the cell (Flamand 1999, Crooks 1994, Blitvich 2001, Schlesinger 1990, Mason 1989, Winkler 1988, 1989). Many forms of this protein have been shown to exist either due to alternative cleavage sites, formation of heterodimers, or differences in glycosylation (Blitvich 1995, 1999, Falgout 1995, Nestorowicz 1994, Mason 1989, Young and Falconar 1989). Previous studies with other flaviviruses containing non-glycosylated forms of the NS1 protein have concluded that glycosylation is not necessary for the dimerization of this protein although the stability of the dimer is reduced (Pryor and Wright 1993, 1994). It has also been noted that dimerization may not be necessary for the secretion of this protein or replication of the virus (Hall 1996).
NS1 is inserted into the endoplasmic reticulum by a hydrophobic signal sequence where it forms a dimer and high mannose glycans are added. The glycosylated protein then proceeds to the Golgi where complex glycans may be added before secretion from the cell (Pryor and Wright 1994, Depres 1991, Flammond 1992, Jacobs 1992, Mason 1989, Post 1991). It has been demonstrated for both DEN and YF viruses that the first glycosylation site (NS1130) is a complex type while the second (NS1207-[DEN]/NS1208 [YF]) is a simple high mannose type (Muylaert 1996, Pryor 1994); however, this mixture is only seen in mammalian cells and not mosquito cells (Blitvich 1999). The lack of complex sugars at the second glycosylation site is hypothesized to be due to this site being buried within the dimer where it cannot be reached for this addition (Hall 1999). Murray Valley encephalitis virus also contains a mixture of complex and high mannose type sugars, the first site (NS1130) is known to be complex type, while the third (NS1207) is high mannose.(Blitvich 2001). The second glycosylation site (NS1175) was not determined in this study.
Studies involving the ablation of the glycosylation sites of the NS1 protein have been performed for other flaviviruses, including DEN and YF viruses (Pryor 1994, 1998, Muylaert 1996, Pletnev 1993, Crabtree 2005). In contrast to WNV, these viruses contain only two glycosylation sites in their NS1 protein; the NS1175 site being absent. Previously, deglycosylation of the NS1 protein of a tick-borne encephalitis virus (TBEV) prM and E genes-containing DEN-4 virus chimera, the NS1130 mutant showed a decrease in neurovirulence while the mutation of the second glycosylation site (NS1207) increased the neuroinvirulence in mice (Pletnev 1993). Similarly, a study of the deglycosylation of YF virus showed that the NS1130 and the combined NS1130/208 glycosylation mutants were attenuated for neurovirulence while the deglycosylated NS1208 mutant alone was not (Muylaert 1996). This study also found that replacement the asparagine of the glycosylation motif with either alanine or serine showed similar results in in vitro studies, namely the lack of the first glycosylation site correlated with a reduction in the rate of RNA synthesis and a delay in the production of infectious virus. Comparable to these data, deglycosylated NS1 of a TBEV/DEN-4 chimera also showed a reduction in infectivity in monkey kidney LLC-MK2 and mosquito C6/36 cell types with the NS1130 mutant showing greater reduction than the NS1207 mutant (Pletnev 1993). Examination of the affects of the deglycosylation of the NS1 protein of DEN-2 virus New Guinea C strain showed that the NS1130 and NS1130/207 mutants had no detectable infectivity titer while the NS1207 mutant had a 100-fold reduction in infectivity titer (Pryor 1998). The NS1207 mutant virus was subsequently examined for mouse neurovirulence at an inoculum of 10 pfu and none of the mice inoculated with the NS1207 mutant virus died while the parental virus caused 75% mortality at this dose. Recently a study involving the deglycosylation of dengue 2 virus strain 16681 showed a decrease in replication of the mutant viruses in C6/36 cells, but not mammalian cells, reduced NS1 secretion from infected cells and attenuation of neurovirulence in mice (Crabtree 2005). This study indicated that the ablation of the NS1207 glycosylation site showed a greater difference than ablation of NS1130 compared to the parental strain.
Another nonstructural protein that may be of interest with regard to flavivirus virulence is the small hydrophobic NS4B protein. The NS4B of West Nile virus (WNV) is cleaved by a combination of viral and host proteases (Chambers et al., 1989; Preugschat et al., 1991) and is believed to associate with other components of the viral replication complex in addition to contributing to evasion of host immune defenses. Within the family Flaviviridae, WNV NS4B exhibits ˜35% identity with other mosquito-borne flaviviruses including yellow fever (YF) virus and members of the dengue (DEN) serogroup. Hepatitis C virus (HCV) NS4B displays negligible amino acid similarity with the WNV protein, however predicted topologies are similar suggesting a common function. Lundin et al., (2003) expressed recombinant HCV NS4B-GFP fusion protein in Hep3B cells and found that it was primarily localized to the endoplasmic reticulum and distributed in a reticular web-like pattern with scattered dense spots thought to represent foci of replication. Accumulation of Kunjin virus NS4B in the perinuclear region has also been described along with induction of membrane proliferation, and there is evidence that NS4B can translocate into the nucleus (Westaway et al., 1997). Recently DEN2 virus NS4B was found to inhibit the interferon-signaling cascade at the level of nuclear STAT phosphorylation (Munoz-Jordan et al., 2004).
A number of publications have described mutations in the NS4B protein in attenuated or passage-adapted mosquito-borne flaviviruses suggesting this protein plays a vital role in replication and/or pathogenesis. It is likely that NS4B interacts with a combination of viral and host factors to allow efficient replication in both vertebrates and mosquitoes. A single coding mutation (P101L) in DEN-4 virus NS4B conferred a small-plaque phenotype in C6/3 6 cells while at the same time increasing plaque size in Vero cells two-fold and Huh7 cells three-fold (Hanley et al., 2003). Pletnev et al. (2002) described DEN4 NS4B T105I and L112S substitutions that occurred in a chimeric virus expressing WNV structural proteins in a DEN-4 virus backbone. Blaney et al. (2003) noted a NS4B L112 F mutation in DEN4 virus passaged in Vero cells. The live attenuated Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 has an 1106V substitution in NS4B (Ni et al., 1995). A viscerotropic Asibi strain of YF virus generated by passaging seven times through hamsters accumulated seven amino acid substitutions including a V981 substitution in NS4B (McArthur et al., 2003). Interestingly YF vaccine strains also display an I95M mutation in NS4B (Hahn et al., 1987; Wang et al., 1995).
While both NS1 and NS4B may play a role in virulence of flavivirus it was previously unclear in the art what changes in these proteins would effectively attenuate flaviviruses. Thus, the present invention answers a long standing need in the art by providing mutant flaviviruses that are high attenuated and identifying mutations in viral nonstructural proteins that can mediate such attenuation.